Rotaviruses (RVs) are major causes of life-threatening diarrheal disease in infants and young children worldwide. Particles are non-enveloped with a complex structure composed of three concentric protein shells that surround the genome of 11 segments of double-stranded (ds) RNA. Similar to influenza viruses, RVs exhibit enormous genetic and strain diversity as a result of point mutations and genetic reassortment between co-circulating strains, and zoonotic infection. How this expanding RV diversity will affect the efficacy of currently available vaccines over time is unclear. In additio to being clinically important, RVs serve as outstanding models to dissect cytoplasmic replication and encapsidation of 11 segments of dsRNA, a puzzle that has fascinated virologists since the first description of these viruses. Our long-term goal is to gain a comprehensive and mechanistic understanding of the elaborate processes that underlie RV replication. Exciting discoveries from our recent work have resulted in several paradigm shifts including i) the VP8* domain of the VP4 spike that mediates cell attachment binds non-sialylated glycans such as histoblood group antigens, (ii) conformational transitions in the VP4 spike mediate virus entry into cells, (iii) serotype-dependent variation occurs in the spike structure, (iv) hitherto unsuspected new enzymatic and RNA binding activities, and conformational switches are present in a nonstructural protein (NSP2) central to RNA replication. In addition, a RV protein (NSP1) with unique properties as an interferon antagonist that is important in host range restriction has been successfully crystallized. This progress forms the foundation for new studies proposed in this competitive renewal application that continue to seek fundamental mechanistic insight into RV biology using a multipronged approach that includes crystallographic and cryo-EM techniques, high- throughput glycan array screening, as well as novel infectivity, enzymatic and functional assays. The specific aims of our proposed work are: (1) to test the hypotheses that genotypic diversity in VP8* of human RVs (hRVs) allows for significant variations in glycan specificity, which may have implications in cell tropism, host specificity, host adaptation and interspecies transmission; and that serotype variations between animal and hRV impact spike structure by determining a subnanometer cryo-EM structure of a globally dominant hRV strain; (2) to examine the hypothesis that the RNA binding multi-functional NSP2 octamer, with built-in structural cues and enzymatic activities, is the master regulator that coordinates viroplasm formation, genome replication/packaging and particle assembly to gain a fundamental understanding of the mechanisms that govern these intricate processes, and (3) to determine the molecular basis for strain-dependent and species-dependent IFN antagonism by RVs, by determining the X-ray structures of two NSP1s representing two different host systems (simian and human). Our structural studies may provide insight into manipulation of the NSP1 gene to design a rationally attenuated, second-generation RV vaccine.